Immunopurification of T-cells from sea bass Dicentrarchus labrax (L.)

The monoclonal antibody DLT15, specific for thymocytes and peripheral T-cells of the teleost fish Dicentrarchus labrax (sea bass), was used to purify immunoreactive cells from blood and gut-associated lymphoid tissue. The purification was performed by immuno-magnetic sorting of leucocyte fractions enriched by Percoll density gradient centrifugation, and the purity of the isolated cells was estimated by cytofluorimetric analysis. Following a single step, the percentage of DLT15-purified cells was 88 +/- 10% for gut-associated lymphoid tissue and 79 +/- 18% for blood leucocytes. DLT15-purified cells from gut-associated lymphoid tissue were employed for RNA extraction and cDNA synthesis. In RT-PCR experiments using as primers degenerate oligonucleotides corresponding to the peptide sequence MYWY and VYFCA of the trout TcR beta chain, a 203 bp product was amplified. When sequenced, the cDNA was found to show 60% nucleotide identity to the trout TcRV beta 3. By 3'-RACE the cDNA was elongated to obtain the TcR constant region, with high similarity to other fish TcR sequences. These results strongly suggest that cells recognised by DLT15 are putative T lymphocytes.     

Differentiation of cells producing polypeptide hormones (ACTH,MSH, LPH, α- and β-endorphin,GH and PRL) in the fetal porcine anterior pituitary

Summary The aim of the present study on the fetal porcine pituitary was (1) to detect by means of the immunoperoxidase technique the earliest stages of cells producing polypeptide hormones: -MSH, ACTH, -LPH, - and -endorphin, growth hormone (GH) and prolactin (PRL), (2) to study the development of the synthesis and the storage of these hormones during fetal life, and (3) to detect whether several hormones can be located in one and the same cell.The corticotropic cells were revealed as the earliest functional elements of the fetal anterior pituitary. Our results indicate clearly that ACTH, -MSH, -LPH, - and -endorphin appear at 34 days in the same regular, round or ovoid cells; no differences in the time of their appearance could be observed. The ACTH-cells, irregular or angular in shape and endowed with cytoplasmic processes such as described in the adult pituitary, were not seen until day 50. The first GH-cells were detected between 40 to 45 days of fetal life. From day 45 to 90, the GH-cells greatly increased in number and in staining intensity of their progressively extending cytoplasmic area, but they displayed the same regular and round shape. The PRL-cells were the last cell type to appear in the fetal pituitary. The first PRL-cells, small in size and round or ovoid in shape with a high nucleus/cytoplasm ratio, were detected at day 70. At day 80, the PRL-cells increased in size and staining intensity. They displayed an irregular elongated or stellated shape and cytoplasmic processes resembling those characteristic of the adult pituitary. These data suggest that in the fetal porcine pituitary: (1) ACTH, -LPH and related peptides are synthesized and stored in the same cells, and (2) PRL and GH appear in individual cellular elements.     

Peptidylglycine α-amidating monooxygenase (PAM) immunoreactivity and messenger RNA in human pituitary and increased expression in pituitary tumours

Bioactivity of many peptides depends upon post-translational -amidation of inactive precursors by two enzyme activities known collectively as peptidylglycine -amidating monooxygenase (PAM). PAM enzymes are particularly abundant in the pituitary. The distribution of PAM immunoreactivity and messenger ribonucleic acid (mRNA) in the adult human pituitary and in pituitary tumours was investigated by use of immunocytochemistry and in situ hybridisation. Immunoreactivity was present in numerous cells of the anterior lobe: staining was intense in a proportion of gonadotrophs and folliculo-stellate cells, but weaker in the majority of somatotrophs and lactotrophs, a few corticotrophs and occasional thyrotrophs. PAM staining was also present in nerves, pituicytes and some endocrine cells within the posterior lobe (the human intermediate zone). Forty pituitary tumours of various types were immunoreactive for PAM; more intensely and uniformly stained than normal anterior lobe. In situ hybridisation with digoxigenin-labelled probes demonstrated intense labelling for PAM mRNA in numerous cells in normal anterior pituitary and in tumours. Many regulatory peptides that require amidation for activity, potential targets for PAM, are present in the pituitary. Many tumour growth factors also require amidation and PAM may regulate these mitogenic peptides in tumours.     

Expression of the cholinergic gene locus in the rat placenta

High amounts of acetylcholine (ACh) and its synthesising enzyme choline acetyltransferase (ChAT) have been detected in the placenta. Since the placenta is not innervated by extrinsic or intrinsic cholinergic neurons, placental ACh and ChAT originate from non-neuronal sources. In neurons, cytoplasmic ACh is imported into synaptic vesicles by the vesicular acetylcholine transporter (VAChT), and released through vesicular exocytosis. In view of the coordinate expression of VAChT and ChAT from the cholinergic gene locus in neurons, we asked whether VAChT is coexpressed with ChAT in rat placenta, and investigated this issue by means of RT-PCR, in situ hybridisation, western blot and immunohistochemistry. Messenger RNA and protein of the common type of ChAT (cChAT), its splice variant peripheral ChAT (pChAT), and VAChT were detected in rat placenta with RT-PCR and western blot. ChAT in situ hybridisation signal and immunoreactivity for cChAT and pChAT were observed in nearly all placental cell types, while VAChT mRNA and immunolabelling were detected in the trophoblast, mesenchymal cells and the visceral yolk sac epithelial cells. While ChAT is nearly ubiquitously expressed in rat placenta, VAChT immunoreactivity is localised cell type specifically, implying that both vesicular and non-vesicular ACh release machineries prevail in placental cell types.     

PEG-mediated plastid transformation: a new system for transient gene expression assays in chloroplasts

Summary Evidence is presented for the introduction of functional copies of the GUS-reporter gene with plastid regulatory signals into chloroplasts after treatment of Nicotiana plumbaginifolia leaf protoplasts with PEG. GUS-activity is found in cells derived from protoplasts treated with PEG in the presence of plasmids harbouring the GUS-gene under the control of plastid promoter and terminator signals (plastid-specific reporter gene constructions). The activity is maintained after chloroplast isolation and incubation with the protease thermolysin under conditions sufficient to completely remove the much higher transient nuclear/cytoplasmic expression of a GUS-gene carrying the CaMV 35S-promoter. Likewise, GUS-activity derived from a plasmid coding for the nuclear/cytoplasmic expression of the reporter gene with a plastid transit presequence is also maintained after these procedures. These results indicate that PEG-treatment is a suitable protocol by which to introduce DNA into chloroplasts for the study of transient gene expression.     

In situ RT-PCR allows the detection of ornithine decarboxylase mRNA in paraffin embedded archival human hyperplastic breast tissues

Expression of ornithine decarboxylase (ODC) is induced by c-Myc oncoprotein and is required for cell proliferation and tumour growth. We have studied the expression of ODC mRNA by in situ hybridisation and in situ RT-PCR in archival human hyperplastic breast tissues. A very low signal was detected by in situ hybridisation, while the in situ RT-PCR on human breast archival tissues demonstrated an over-expression of ODC mRNA in epithelial cells characterised by some degree of hyperplasia, maintaining the morphology of the archival tissue intact despite the multiple steps of fixation, permeabilization and thermal cycling.     

Antifibrotic effects of <Emphasis Type="Italic">Salvia miltiorrhiza </Emphasis>on dimethylnitrosamine-intoxicated rats

Excessive oxidative stress is implicated in hepatic fibrogenesis. Extracts of Salvia miltiorrhiza (Sm) have been shown to protect cells against oxidative stress. In this study we investigated the in vitro and in vivo effects of Sm on hepatic fibrosis. A cell line of rat hepatic stellate cells (HSC-T6) was stimulated with transforming growth factor-1 (TGF-1). The inhibitory effects of Sm (50~400 g/ml) on TGF-1-induced -smooth muscle actin (-SMA) secretion and the mRNA expressions of fibrosis-related genes, including -SMA, connective tissue growth factor (CTGF), and tissue inhibitor of metalloproteinase-1 (TIMP-1), were assessed. Fibrosis was induced by dimethylnitrosamine (DMN) administration in rats. DMN-treated rats were randomly assigned to 1 of 4 groups: saline, Sm (20 mg/kg), Sm (100 mg/kg), or silymarin (100 mg/kg), each given by gavage twice daily for 5 weeks starting from the onset of DMN administration. Sm (200 and 400 g/ml) significantly inhibited TGF-1-stimulated -SMA secretion and the mRNA expressions of -SMA, CTGF, and TIMP-1 in HSC-T6 cells. Fibrosis scores of livers from DMN-treated rats with either a low (1.8 ± 0.2) or high (1.8 ± 0.1) dose of Sm, or silymarin (1.4 ± 0.2) were significantly reduced in comparison with DMN-treated rats receiving saline (3.1 ± 0.1). Hepatic collagen contents were also significantly reduced by either Sm or silymarin treatment. The mRNA expression levels of -SMA, TGF-1, and procollagen I were all attenuated in Sm- and silymarin-treated rats. Moreover, levels of plasma aspartate transaminase activities were reduced by Sm and silymarin treatment. In conclusion, our results show that Sm exerted antifibrotic effects in both HSC-T6 cells and in rats with DMN-induced fibrosis.     

Distribution of β2-like adrenergic receptors in the cnidarian Renilla koellikeri as revealed by autoradiography and in situ hybridization

Autoradiography and in situ hybridization were used to examine the histological distribution of the previously characterized 2-like adrenergic receptors involved in the bioluminescent activity of the sea pansy Renilla koellikeri. The use of [³H]-(±)CGP12177 as radiologand revealed autoradiographic labelling of the refringent granule-filled endoderm at the base of autozooid tentacles and autozooid columns, and in the corresponding endoderm of siphonozooid polyps, all areas where photocytes are concentrated. The presence of excess (10 M) unlabelled (±)CGP12177 or atenolol in the incubation mixture substantially reduced total [³H]-(±)CGP12177 labelling. Under low stringency hybridization washing, human 2-adrenoceptor oligonucleotide probe signals were detected in granular cells located in those areas of polyp endoderm that were labelled by [³H]-(±)CGP12177. These cells were previously shown to be distinct from, but in close proximity to photocytes. No other cell or tissue type was labelled in polyps or throughout colonial tissues. The results suggest that a conserved form of 2-adrenergic receptors is present and synthesized in a unique type of endodermal cell indirectly involved in sea pansy bioluminescence control.     

Metabolism of 18β-glycyrrhetinic acid in Sphingomonas paucimobilis strain G5

Seven strains capable of utilizing 18-glycyrrhetinic acid (18-GRA) as a sole carbon and energy source were isolated from soil samples by enrichment culture technique. One of these strains, named strain G5, was identified as Sphingomonas paucimobilis. When this strain grew on 18-GRA, several metabolites were detected in the culture broth. A major metabolite, tentatively named M-A, was isolated and its chemical structure was determined by nuclear magnetic resonance (NMR) and gas chromatography-mass spectrometry (GC-MS).     

Catecholaminergic nerves in the embryonic chick ovary: co-localization with β2-adrenoceptor-bearing steroidogenic cells

Summary The present study investigates the innervation of the embryonic chick ovary with regard to (i) development and compartmentalization of catecholaminergic nerves, and (ii) presence of adrenoceptors on steroidogenic target cells of catecholaminergic nerve terminals. Catecholaminergic nerve fibers visualized by glyoxylic acid-induced histofluorescence first appeared at embryonic day (E) 13. From E15 through E21 the density of fluorescent aminergic nerves increased markedly in parallel with the concentration of catecholamines and numbers of nerve bundles and single axons seen at the electron-microscopic level. Catecholaminergic nerves were confined to the ovarian medulla and closely associated with interstitial cells. Nerve terminals approached interstitial cells up to a distance of 20 nm and, in their majority, exhibited uptake of the false adrenergic transmitter 5-hydroxydopamine. Although adrenaline amounted to 14% of the total catecholamine content at E21, adrenaline immunoreactivity was only detected in adrenal chromaffin cells, but not in nerve fibers or cell bodies within the ovary. Interstitial cells structurally matured between E15 and E21 as documented by an increase of smooth endoplasmic reticulum and tubular mitochondria. Monoclonal antibodies mAB 120 and BRK 2 raised against avian ₁ and mammalian ₂-adrenergic receptors revealed the presence of ₂-adrenoceptor-like immunoreactivity on the surface of interstitial cells, but not on any other cell type.The results are consistent with the notion of a dense adrenergic innervation of the embryonic chick ovarian medulla and its steroidogenic interstitial cells, and suggest the chick ovary as an excellent model for elucidating the functional role of a neural input to steroidogenic cells during development.     

Establishment and characterization of a clonal human T-cell line, MKB-1 derived from a patient with acute myeloblastic leukemia

A human T-cell line, designated as MKB-1, was established by cloning procedures in a suspension culture from a peripheral blood of a 17-year-old female patient with acute myeloblastic leukemia. The immunological marker profile of MKB-1 indicated that unlike a myeloid phenotype of the original leukemic cells, the cells were positive for CD3 (both cell surface and cytoplasm), T cell receptor (TcR) alpha/beta heterodimer, CD4, CD5, CD7, CD10, CD57 (Leu7), SN-1 and the cytoplasmic TcR beta chain. These findings indicate the T cell nature of the established cells. Terminal deoxynucleotidyl transferase (TdT) was also detected in 60%. We did not detect markers of human myeloid and B cell associated antigens, HLA-class II or immunoglobulin chains. Cytogenetic study revealed that the MKB-1 cells had a female hypo-tetraploid karyotype with chromosomal abnormalities including a translocation between chromosomes 10 and 14. The breakpoint of chromosome 14 of this translocation, 14q11.2, is known to be the location of TcR alpha and delta genes; t(10; 14) (q26; q11.2) is a variant type of a T cell neoplasm-associated translocation, t (10; 14) (q24; q11.2). The MKB-1 cell line is unusual in that its T cell characteristics are phenotypically and cytogenetically distinct from the original myeloid leukemia cells.     

Immunohistochemical evidence identifying the site of androgen production in the ovary of the protogynous grouper <Emphasis Type="Italic">Epinephelus merra</Emphasis>

Androgen plays an important role in the developing ovaries of female fish. However, little is known regarding either the sites of production of androgen or its functional roles. In the present study, we investigated immunohistochemically the localization of cholesterol-side-chain-cleavage (P450scc) and cytochrome P45011-hydroxylase (P45011) with antibodies P450scc and P45011 in the ovary of the female honeycomb grouper Epinephelus merra during its reproductive cycle. Clusters of strongly immunopositive cells, with 100–1000 cells in each cluster, against both P450scc and P45011, were observed throughout the annual reproductive cycle in tissue near blood vessels in the tunica ovary surrounding the outer periphery of the ovary. The ultrastructural characteristics of these cells showed that they were steroid-producing cells. In contrast, immunopositive cells against P450scc but not against P45011 were localized in the theca layer surrounding the outer periphery of oocytes. These results suggest that two distinct steroid biosynthesis sites exist in the ovary and that cells at the two sites differ functionally. The only cells that biosynthesize 11-ketotestosterone are found in clusters in the vicinity of blood vessels; they possibly play a physiological role in oocyte growth and gonadal restructuring during the sex change of individuals of this species. CREST, JST (Japan Science and Technology Corporation), and the Takeda Science Foundation provided grants supporting this work. This study was also partially supported by a grant for the 21st century COE project entitled, The Comprehensive Analyses of Biodiversity in Coral Reef and Island Ecosystems in Asian and Pacific Regions. We also gratefully acknowledge a Japanese Government (Monbukagakusho) Scholarship.     

β-carotene is the intermediate exported from the chloroplast during accumulation of secondary carotenoids in Haematococcus pluvialis

Recent findings on the localization in Haematococcus pluvialis of anearly and a late enzyme in astaxanthin synthesis have suggested thesequential involvement of two compartments, the chloroplast (site of earlysynthesis) and the cytoplasmic lipid vesicles. Here the intermediatetransported across the chloroplast envelope into the cytoplasm is elucidatedby means of the inhibitors diphenylamine (DPA) and2-(4-chlorophenylthio)-triethylamine (CPTA). Based on the amounts of theintermediates accumulated upon inhibitor treatment and their occurrencein the lipid vesicle fractions, it is concluded that the transport takes placeafter -ionone ring cyclization. A non-vesicle mechanism of -iononecarotenoid transport from the chloroplast towards the cytoplasmic lipidvesicles via carotenoid binding proteins is suggested.     

Acidophilic granulocytes of the marine fish gilthead seabream (<Emphasis Type="Italic">Sparus aurata</Emphasis> L.) produce interleukin-1β following infection with<Emphasis Type="Italic"> Vibrio anguillarum</Emphasis>

The fish immune response to Gram-negative bacteria is poorly understood. In this study, we use a monoclonal antibody (mAb) specific to acidophilic granulocytes from the marine fish gilthead seabream (Sparus aurata L.), together with an antiserum specific to interleukin-1 (IL-1) from this species, in order to investigate whether these cells are involved in the immune response against the pathogenic bacterium Vibrio anguillarum and, in particular, in the production of the pro-inflammatory cytokine IL-1. We found that gilthead seabream head-kidney, peritoneal exudate and peripheral blood leukocytes accumulated proIL-1 intracellularly when challenged in vitro with V. anguillarum, whereas only peritoneal exudate and blood leukocytes were able to accumulate proIL-1 following infection. Importantly, the blood leukocytes from infected animals that accumulated proIL-1 were shown to be the acidophilic granulocytes. A rapid mobilization of such cells from the head-kidney to the site of inflammation following infection with V. anguillarum was also observed. This work was supported by the Spanish Ministry of Science and Technology (grants BIO2001-2324-C02-02 and AGL2002-03529, and fellowship to J. García-Castillo), Spanish Ministry of Education, Culture and Sport (fellowship to E. Chaves-Pozo) and Fundación Séneca, Coordination Centre for Research (grant PI-51/00782/FS/01 and fellowship to P. Pelegrín).E. Chaves-Pozo and P. Pelegrín contributed equally to this work.